Fibrinogen is one of the plasma glycoproteins mainly produced by liver (parenchymal) cell, and is a macromolecular glycoprotein having two each of 3 different kinds of polypeptide chains called Aα chain, Bβ chain and γ chain (6 chains in total). The molecular weight of each polypeptide chain is about 67,000 for Aα chain, about 56,000 for Bβ chain, and about 47,500 for γ chain, and a complete fibrinogen molecule composed of these chains associated via a disulfide bond has a molecular weight of about 340,000 (patent document 1).
Fibrinogen is an important protein present in normal plasma at 2-3 g/L, which exhibits defense and hemostasis functions in living organisms by inducing, when tissues are damaged, adhesion of platelets to the wound and subsequent hematological gelation. Therefore, when the fibrinogen level in blood decreases due to massive bleeding, severe infections and the like, the hemostasis mechanism collapses and bleeding cannot be suppressed to cause bleeding tendency, which is life-threatening.
Fibrinogen preparation is effective for preventing serious bleeding by increasing the fibrinogen concentration in blood by intravenous administration and the like, and is widely applied to a replacement therapy of congenital and acquired fibrinogen deficiency and the like. Also, fibrinogen is widely used as a main component of fibrin adhesive to be used for adhesion and closing of tissue during surgery.
At present, fibrinogen used as pharmaceutical products is mainly prepared from human pooled plasma collected from an unspecified large number (several thousand or more) of blood donors, and subjected to various pathogens inactivating, removing methods such as a tri-n-butyl phosphate (TNBP)/polysorbate 80 treatment, a filtration treatment with a virus removal membrane, a heat treatment and the like in an attempt to eliminate the risk of contamination with infectious agents such as hepatitis virus (e.g., HCV and the like), immunodeficient virus (e.g., HIV and the like), abnormal prion and the like. However, no matter how much the safety measure is taken, the risk of disease transmission due to the use of blood as a starting material cannot be eliminated completely. When a fibrinogen preparation derived from human pooled plasma is used, therefore, it is necessary to consider the effect provided and the risk of disease transmission and the like, sufficiently study the need thereof, use only in the minimum necessary amount, and sufficiently observe the progress after administration. Furthermore, since human pooled plasma is mainly supplied by blood donation, stable supply of fibrinogen in the future is also questioned.
To solve these problems, production of fibrinogen by utilizing a gene recombination technique has been tried. However, fibrinogen has not been placed in the market as a recombinant pharmaceutical product, even though plasmaproteins such as factor VIII, factor IX, albumin and the like have already been placed in the market as recombinant pharmaceutical products.
One of the causes preventing the development is the fact that fibrinogen is a huge protein molecule having an association of 6 polypeptide chains, and a functional fibrinogen molecule is difficult to produce even when 3 proteins of fibrinogen Aα chain, Bβ chain and γ chain are simultaneously expressed in Escherichia coli, and that a functional fibrinogen molecule can be produced in yeast and animal cells but production in a sufficient amount is not attainable, which in turn prevents practicalization from the aspect of production cost (non-patent document 1, patent documents 1 and 2).
Another cause is the fact that, when fibrinogen is expressed and cultured in animal cells, degradation of fibrinogen markedly progresses in the later stage of culture. Generally, cultured cells grow in the order of lag phase, logarithmic growth phase, stationary phase, and death phase, and the number of cultured cells and the production amount of recombinant protein are correlated. Therefore, when a recombinant protein is produced, it is considered that an extension of the period of stationary phase when the number of cultured cells reaches the peak, i.e., the later stage of culture, leads to an increase in the production amount of the recombinant protein. Remarkable degradation of fibrinogen in the later stage of culture is a fatal problem for mass production of fibrinogen, and renders the production of high quality recombinant fibrinogen in a high yield even more difficult.
Plasmin is a serine protease that hydrolyzes fibrinogen and fibrin produced from fibrinogen (dissolve of fibrin: fibrinolysis). In the fibrinolytic system, plasminogen is restrictively degraded by a plasminogen activator to be a plasmin having an enzyme activity, which shows a function to mainly dissolve fibrin thrombus.
An α2 plasmin inhibitor (α2PI) is a main inhibitory factor of plasmin which takes charge of the fibrinolytic system, and is a protein that specifically binds to plasmin at a ratio of 1:1 to form a plasmin-α2PI complex (PIC), which rapidly deactivates the plasmin activity.
Plasminogen activator inhibitors (PAI)-1 and PAI-2 are inhibitors belonging to the serine protease inhibitor superfamily (SERPIN) and present in vivo. It is a protein that suppresses generation of plasmin from plasminogen by inhibiting the plasminogen activator. It has been reported that PAI-1 is present in normal plasma at a concentration of about 20 ng/mL, whereas PAI-2 is generally undetectable in non-pregnant plasma (non-patent document 2).
However, the relationship between α2PI and/or PAI-2, and a fibrinogen production-enhancing effect in recombinant fibrinogen producing cells has not been reported at all, and the effect thereof is unknown.